Analysis and purification of polypeptides from biological samples often involves the lysis of a cell or tissue sample as a precursor to downstream polypeptide processing. It is well recognized that genomic DNA is often released during cell or tissue lysis and that this causes an increased viscosity of the lysate that can interfere with downstream polypeptide analysis or purification. Common biotechnical methods that require or benefit from reduction in lysate viscosity include:
1) Western blot analysis. For example, Chen et al., Front. Biosci., 2014, 4, 2365-2377 describes the process of reducing lysate viscosity by several pulses of sonication for the preparation of lysates destined for Western blot analysis (see, Section 3.5. Sample preparation).
2) Immunoprecipitations. For example, Moser et al., Nat. Protoc., 2009, 4, 674-685 describes the critical nature of reducing lysate viscosity by passing four times through a syringe fitted with a 21 G 1.5 inch needle. It is also noted that the gauge of the needle is critical to successful lysis, viscosity reduction and immunoprecipitation step (see, Procedure step 7).
3) Laboratory and industrial scale protein purification. For example, Maine et al., Nat. Protoc., 2010, 5, 1447-1459 states that “proper sonication is mandatory because otherwise the high viscosity of the lysate will prevent it from being handled through the initial precipitation” (see, Procedure, Step 10 p. 1452). Another example can be found in DeWalt et al., Protein Expression & Purification, 2003, 28, 220-223 describes a method of reducing a microbial lysate viscosity by utilizing a polycationic compaction agent such as spermidine to facilitate the downstream purification of a His-tagged protein from the treated lysate.
Many methods have been devised that remove or shear genomic DNA and thereby reduce lysate viscosity to allow for more efficient downstream processing of lysate polypeptides. One method is sonication (see, Maine et al., supra), which effectively breaks up genomic DNA but can often result in partial or total denaturation of the target protein. Another method is passage of the lysate through a fine gauge needle (see, Moser et al., supra). This method is very time consuming and tedious and does not scale well to larger lysate volumes. Yet another method includes treatment of lysates with a compaction agent (see, DeWalt et al., supra). Yet another method involves treating the lysate with nucleases such as Benzonase® (see, U.S. Pat. No. 5,173,418). Several methods have been reported that use a filter system to reduce viscosity of a cell or tissue lysate. For example U.S. Patent Application Publication No. 2012/0053328 reports a filter system that retains genomic DNA while allowing polypeptides to be collected in the filtrate. This is a relatively rapid and gentle method, however the fact that it requires the use of specialized equipment such as a centrifuge or a vacuum limits its usefulness. Chromosomal integration of nuclease genes into protein expression systems have been used to reduce viscosity (see, Boynton et al., Applied & Env. Microbiol., 1999, 65, 1524-1529). In addition, treatment of lysates with exogenous nucleases, sonication of lysates (see, Methods in enzymology Ed. J. Lorsch. 2015. Strep-tagged protein purification. vol. 559 chapter 5 p. 61) have also been used to reduce viscosity of lysates.
Methods for rapid, gentle, and one-step removal of genomic DNA and cellular debris from crude cell and tissue lysates that does not require the use of specialized equipment while allowing for collection of cellular polypeptides from, for example, viscous cell lysates is desired.